Thirty-two, timed-pregnant Sprague-Dawley rats were housed in an uncrowded, quiet animal facility room on a 12 h light/dark cycle and were provided with food and water ad libitum. Parturition was checked daily, and the day of birth was considered postnatal day (P) 0. On P2, all pups were briefly gathered, and 10 pups assigned at random to each dam. They experienced one of the following early-life rearing conditions: (a) Brief separation from the dam (augmented care, or augmented-experience'), which took place daily from P2 to P8. (b) Undisturbed controls, that remained in cages that were not touched between P2 to P8. Rats were either sacrificed on P9, between 08:00 and 12:00 or maintained under standard animal facility rearing conditions, weaned on P21, and grown, 3-4-per cage into adulthood. All experiments were approved by the University Animal Care Committee and conformed to National Institutes of Health guidelines.
Augmentation of maternal care via brief daily separation, and observation of maternal care
Cages were brought into laboratory daily at 8:30 AM. The dam and the pups were placed into separate bedded cages (pups were kept euthermic via a heating pad located underneath the cage). After 15 min, pups were placed back into their home cage, followed by the dam, and returned to the vivarium. Undisturbed litters remained in the vivarium from P2 to P9. For all experimental groups, cage change did not occur during this time. After pups and were returned to the vivarium, maternal behavior, (including licking and grooming, was observed using a protocol based on (Liu et al., 1997
) as used by (Fenoglio et al., 2006
). Maternal behavior was also determined in undisturbed litters. Each maternal observation session consisted of 10 epochs of 3 min each. Within each epoch, the duration of licking and grooming of pups was recorded during the first min. The total amount of time spent licking and grooming was scored per session, added for all the sessions and multiplied by three to provide the total amount of maternal care during the 30 min session.
In situ hybridization (ISH) for CRH mRNA
Rats were rapidly decapitated at P9 (n=5 for control and n=6 for experience-augmented pups) and between P45 and P60 (n=4 /group), and the brains were quickly dissected and frozen in dry ice. Brains were sectioned at 20 μm using a cryostat, collected onto gelatin coated slides and stored at -80 °C. ISH histochemistry was conducted as described previously using deoxy-oligonucleotide probes (Avishai-Eliner et al., 2001b
). Probe was labeled with 35
S using routine terminal deoxynucleotide transferase methodology. Briefly, sections were brought to room temperature, air-dried and fixed in fresh 4% buffered paraformaldehyde for 20 min, followed by dehydration and rehydration through graded ethanols. Sections were exposed to 0.25% acetic anhydride and 0.1 M triethanolamine (pH 8) for 8 min and were dehydrated through graded ethanols. Pre-hybridization and hybridization steps were performed in a humidified chamber at 42 °C in a solution of 50% formamide, 5× SET, 0.2% SDS, 5× Denhart's, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, 100 mM dithiothreitol and 10% dextran sulfate. Following a 1 h prehybridization, sections were hybridized overnight with 0.25×106
CPM of labeled probe. After hybridization, sections underwent serial washes at 42 °C, most stringently at 0.3× SSC for 30 min at room temperature. Sections were then dehydrated through increasing ethanol concentrations, air-dried and apposed to film (Kodak BioMax MR Film, Eastman Kodak Co. NY, USA), for 5–10 days.
ISH signal was analyzed on scanned, digitized PVN images of sections at coronal levels corresponding at 3.8-3.5 mm anterior to bregma. The Image Tool software program (UTHSC, San Antonio, TX) was used (Fenoglio et al., 2006
) after determining the linear range of optical densities (ODs) using 14
C standards. Values were only used if they fell in the linear range and were corrected for background by subtracting the OD of signal over the thalamus. OD from two optimal sections was averaged to generate an expression value for each PVN, which is expressed in nCi/g. These values were then used to calculate group means± standard error of the mean (SEM).
Immunocytochemistry (ICC) studies
At P9 or at P45 rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then perfused through ascending aorta with 0.9% saline solution, followed by freshly prepared, cold 4% PFA in 0.1 M sodium phosphate buffer (PB) pH 7.4, cryoprotected in 15% and 30% sucrose/PB solution, and stored at -80°C. Brains were sectioned at 30μm using a cryostat and sections were collected as four series with 90μm intervals in between sections in tissue-culture wells containing 0.1 M PB. Every fourth matched section was subjected to ICC. For each experiment, sections of experience-augmented and control rat brains were processed concurrently in parallel wells. For single labeling CRH ICC, free-floating sections of experience-augmented (n=5) and control (n=5) rats were subjected to standard avidin-biotin complex (ABC) methods (Fenoglio et al., 2006
). Briefly, after several washes with 0.01 M phosphate-buffered saline (PBS) containing 0.3% Triton X-100 (pH 7.4, PBS-T), sections were treated for 30 min in 0.3% H2
in PBS, followed by blockade of non-specific sites with 1% bovine serum albumin (BSA) in PBS for 30 min and incubation for 48 hours at 4°C with anti-CRH (1:40,000; gift from Dr. W.W. Vale, Salk Institute, La Jolla, CA, USA) in PBS After 3 times 5 min washes in PBS-T, sections were incubated in biotinylated rabbit-anti-goat IgG (1:200, Vector, Burlingame, CA) in PBS containing 1% bovine serum albumin for 2 hours at room temperature. After washing in PBS-T (3 × 5 min), sections were incubated in ABC solution (1:100; Vector) for 2 hours at room temperature. Sections were then rinsed again in PBS (3 × 5 min). The reaction product was visualized by incubating sections for 8-10 min in 0.04% 3,3-diaminobenzidine (DAB) containing 0.01% H2
All counts were performed without knowledge of experimental group (blindly). CRH immunopositive cells were visualized using a Nikon E400 microscope, and counted in anatomically matched sections using systematic sampling methods: Briefly, CRH immunopositive cells were counted in four sections which were 90μm apart per animal. We obviated double counting by focusing throughout the 30μm section and including a cell only at the level where it had a clear complete nuclear profile. Because the diameter of the CRH positive cell nucleus was considerably smaller than the thickness of each cryostat section, each cell was counted once only. The PVN and ACE were sampled at coronal levels 3.8-3.5 mm anterior to bregma, and the BnST at levels corresponding to 5.0-4.7mm anterior to bregma. The intensity of CRH immunoreactivity was analyzed on photomicrographs taken through a digital camera (Spot Digital camera, RT color V3.0, Diagnostics Instruments MI, USA) with standardized light source and standardized exposure. Analyses were carried out using ImageJ (version 1.41, NIH, Bethesda, MD, USA). Densities are expressed in OD units after correcting for background by subtracting the density of the immunoreactive signal over the anterior commissure. Numbers of CRH immunopositive neurons and CRH OD from four sections were averaged. The numbers of cells/section and OD/section were used to calculate group means ± SEM.
For double labeling ICC of CRH and vGlut2 (the predominant vGlut isoform in the hypothalamus; Herzog et al., 2001
), sections of experience-augmented and control rats (n = 3/group) were washed 3×10 min in 0.01 M PBS, and treated with 1% Triton X-100 in 0.01 M PBS, for 30 min. Sections were placed in 2% normal goat serum and incubated for 48 hours at 4°C in a mixture of rabbit anti-CRH serum (1:10,000) and guinea pig anti-VGlut2 serum (1:10,000; AB5907; Chemicon). After three, 10 min washes in 0.01 M PBS, sections were incubated in a secondary antiserum cocktail (1:400; goat-anti-rabbit IgG 568 and goat-anti-guinea pig IgG Alexa Fluor 488; Molecular probes, Eugene, Orgeon).
The number of vGlut2 boutons contacting PVN-CRH neurons was assessed by confocal laser-scanning microscopic analysis (n=66 cells for control and 76 cells for experience-augmented rats). Images of the parvocellular division of the PVN (at coronal levels 3.8-3.5 mm anterior to bregma) were collected using a Zeiss LSM510 confocal scanning system. Five, 2 μm thick optical sections were collected along the Z-axis throughout the thickness of the whole neuron Images were acquired with excitation wavelength of 488nm (green) and 543nm (red) with a 40× oil objective employing the minimum pinhole size. Images were imported as TIFF files at the resolution of 1024×1024 pixels. vGlut2 boutons contacting PVN-CRH neuron were counted through the complete Z-axis in each optical section, and averaged (vGlut2 boutons/optical section/CRH cell). A vGlut2 bouton was considered to be apposed to the CRH cell body only when there was no visible space between the CRH cell membrane and the bouton. In addition cell size was measured in the central optical section for each CRH neuron using Zeiss LSM Image browser software (version 220.127.116.11). Analyzed cells were selected based on the following criteria: 1) CRH immunopositive; 2) fully visible soma within the Z-stack; 3) a clearly identifiable nucleus. After all of the quantitative analyses were completed, images used for illustration were optimized for brightness and contrast using Adobe Photoshop 7.0.
Samples consisted of micro-punched PVN (Palkovits, 1973
) or dissected thalamus from an individual rat. Rats were rapidly decapitated at P9 (n=12 per group) and at P45 (n=5 per group), and the brains were quickly dissected and frozen on powdered dry ice. The PVN was punched out (needle gauge 21) from consecutive 150μm thick cryostat sections. Tissue was homogenized in 1.5 ml eppendorf tubes in ice cold 0.32 M sucrose, 0.1 M Tris–HCl (pH 7.4) containing a protease inhibitor cocktail (PIC, Complete™; diluted according to manufacturer's instructions; Roche, Alameda, CA). Protein concentration was determined (Bio-Rad, Hercules, CA), and equal amounts of protein were diluted in Laemmli buffer, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and visualized using the enhanced chemiluminescence (ECL)-Plus kit (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (Brewster et al., 2005
). Briefly, 2 μg of protein extracts were separated on a 10% SDS-PAGE and transferred to Hybond-P polyvinyl difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 10% nonfat milk in 1× PBS overnight at 4 °C and probed with guinea pig anti-vGlut2 (1:800,000, AB5907; Chemicon, Temecula, CA), or rabbit anti-vGat (1:150,000 generous gift of Dr. Edwards UCSF, San Francisco, CA, USA), or rabbit anti-NRSF (1:20,000, H-290-sc25398; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in each case combined with rabbit anti-actin (1:60 000; Sigma, St Louis, MO). Following washes in PBS-1% Tween (PBS-T) (3 × 5 min), membranes were incubated with secondary antibodies made in donkey, conjugated to horseradish peroxidase (1:10 000) in 5% non-fat milk in PBS for 1 h at room temperature. Membranes were washed in PBS-T (3 × 5 min) and incubated with ECL-Plus. Immunoreactive bands were visualized by apposing membranes to Hyperfilm™ ECL (Amersham Pharmacia Biotech). PVN and thalamic extracts of individual rats of different groups were run concurrently on the same gel. Specificity of signal was examined by excluding the primary antibodies in the presence of the secondary antibodies. These treatments resulted in no immunoreactive-vGlut2, vGat or NRSF bands.
Western blot data acquisition and analysis were accomplished by measuring OD of immunoreactive bands from the ECL films using ImageTool. Several durations of exposure were carried out, to ascertain that optical densities were in a linear range. The OD of vGlut2, vGat and NRSF bands was normalized to β-actin of the same sample. Individual OD's were normalized to the mean value of the control OD and used to generate mean values / group ± SEM.
Electron microscopic immunostaining
P9 Rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then perfused through the ascending aorta with 0.9% saline solution, followed by freshly prepared, cold 4% PFA; 15% picric acid and 0.08% glutaraldehyde in 0.1 M PB, pH 7.4. Brains were removed and postfixed in 4% PFA overnight and stored in 0.1M PB. Brains were sectioned at 30μm using a vibratome (Lancer) and sections were collected in multi-well plates containing 0.1 M PB. For CRH ICC, free-floating sections of experience-augmented (n=5) and control (n=5) rats were processed according to standard avidin-biotin complex (ABC) methods with minor adaptations for the electron microscopic staining. Specifically, sections were treated for 30 min in 0.3% H2O2 in PBS. Sections were incubated for 1 hour in freezing solution (0.1M PB, 25% sucrose; 10% glycerol) followed by freeze/thaw in liquid nitrogen repeated 3 times. Then, sections were washed several times with 0.01 M PBS followed by incubation in 1% BSA in PBS for 30 min to block non-specific staining. After a 10-minute rinse in PBS, sections were incubated for 48 hours at 4°C with anti-CRH (1:40,000) in PBS containing 1% bovine serum albumin and washed 3 times in PBS-T, 5 min each. Subsequently, sections were incubated in biotinylated rabbit-anti-goat IgG (1:200, Vector, Burlingame, CA) in PBS for 2 hours at room temperature. After washing in PBS (3 × 5 min), sections were incubated in ABC solution (1:100; Vector) for 2 hours at room temperature. Sections were then rinsed again in PBS (3 × 5 min). The reaction product was visualized by incubating sections for 8-10 min in 0.04% 3,3-diaminobenzidine (DAB) containing 0.01% H2O2. Sections were then osmicated (1% OsO4 in PB) for 30 min, dehydrated through increasing ethanol concentrations (using 1% uranyl acetate in the 70% ethanol for 30 min), and flat embedded in Durcupan between liquid release-coated slides and coverslips (Electron Microscopy Sciences, Fort Washington, PA) followed by capsule embedding. Blocks were trimmed and ribbons of serial ultrathin sections were cut (using a Leica Ultracut E), collected on Formvar-coated single slot grids and examined under an FEI Biotwin electron microscope.
The quantitative and qualitative analysis of synapse number was performed in a blinded fashion. To obtain a complementary measure of axosomatic synaptic number, unbiased for possible changes in synaptic size, the dissector technique was used. On consecutive 90-nm-thick sections we determined the average projected height of the synapses and used about 30% of this value as the distance between the dissectors. On the basis of this calculation, the number of axosomatic synapses was counted in 2 consecutive serial sections about 270 nm apart (termed reference and look-up sections) of 10 CRH-immunolabeled perikarya profiles in each animal. Synapse characterization was performed at a magnification of 20,000. Symmetric and asymmetric synapses were counted on all selected neurons only if the pre- and/or postsynaptic membrane specializations were seen and synaptic vesicles were present in the presynaptic bouton. Synapses with neither clearly symmetric nor asymmetric membrane specializations were excluded from the assessment. The plasma membranes of selected cells were outlined on photomicrographs, and their length was measured with the help of a cartographic wheel. Plasma membrane length values measured in the individual animals were added, and the total length was corrected to the magnification applied. Synaptic densities were evaluated according to the formula NV = Q−/Vdis, where NV represents number per volume and Q– represents the number of synapses present in the reference section that disappeared in the look-up section and Vdis is the dissector volume (volume of reference), the area of the perikarya profile multiplied by the distance between the upper faces of the reference and look-up sections (i.e., the data are expressed as numbers of synaptic contacts per unit volume of perikaryon). Section thickness was determined using the Small's minimal fold method. The synaptic counts were expressed as numbers of synapses on a 100μm membrane length unit.
Whole cell recordings were made from presumed CRH neurons from the parvocellular paraventricular hypothalamic nucleus. Rats were rapidly decapitated at P9 (n=8 rats per group), and at P30 (n=3 rats per group) brains quickly dissected and 300μm hypothalamic slices cut using a vibratome. Hypothalamic slices were maintained at 33 °C and perfused with ACSF. The bath solution (ACSF) consisted of 124mM NaCl; 3mM KCl; 2mM CaCl2; 2mM MgCl2; 1.23mM NaH2PO4; 26mM NaHCO3; 10mM glucose pH=7.4 with NaOH and was continuously bubbled with 5% CO2 and 95% O2. The patch pipettes were made of borosilicate glass (World Precision Instruments) with a Sutter micropipette puller (PP-97). The tip resistance of the recording pipettes was 4-6 MΩ after filling with a pipette solution containing 145mM KMeSO4, 1mM MgCl2; 10mM Hepes; 1.1 mM EGTA; 2mM Mg-ATP; 0.5 mMNa2-GTP pH=7.3 with KOH and 0.1% Lucifer Yellow (LY). After a gigaohm seal and a whole cell access were achieved, the series resistance was between 20 and 40 MΩ and partially compensated by the amplifier. Both input resistance and series resistance were monitored throughout the experiments. mEPSCs and mIPCSs were recorded under voltage clamp in the presence of TTX and bicuculline, with pipette solution containing KMeSO4 (for mEPSCs) or TTX and CNQX plus AP-5, with pipette solution containing KCl (for mIPSCs) with a multiclamp 700A amplifier (Axon Instruments, Inc). The use of KCl significantly increases the conductance of GABA-A receptors and makes recording a mIPSCs easier. PVN cells were held at −60 mV. Detection of mEPSC and mIPSC events were performed offline with the software Axograph 4.9 (Axon Instruments, Inc.). Frequency and Amplitude of mEPSCs (or mIPSCs) were generated after detection of mEPSC events, as described previously (Gao and van den Pol, 2001
). Putative CRH neurosecretory parvocellular PVN neurons were identified on the basis of their visualized position within the PVN and based on the shape of the neuron.
Immunocytochemical identification of CRH neurons
After recording with electrodes containing LY in the pipette solution, slices were postfixed overnight in 4% paraformaldehyde at 4 °C overnight, then washed in 0.1 M PB, embedded in agar and sliced with vibratome to 50 um thick sections. After washes in PBS-T, sections were incubated with rabbit anti-CRH (1:10,000) in 1% BSA in PBS-T for 48h at 4°C. After 3 5 min washes in PBS-T, goat anti-rabbit antibody (Alexa Fluor 568 1:400) in 1% BSA in PBS-T was applied for 3 h to label CRH neurons. Sections were then sealed on glass slides with Vectashield to avoid bleaching, then examined with a fluorescent microscope (Zeiss Axiophot microscope). Overlap of LY images with Alexa Fluor 568 staining was taken as identification of CRH neurons; however leakage of cell contents including the LY in the slices impeded positive identification of the recorded neurons. Therefore, we considered for analysis only recorded neurons residing in the CRH-rich, dorsomedial parvocellular field of the PVN.
Chromatin Immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) assay was based on a fast ChIP protocol (Nelson et al., 2006
). Brains were dissected from P12 rats, hippocampi isolated and frozen in tubes on dry ice, and stored at -80°C until use. All ChIP buffer solutions contained Protease inhibitor complex (Roche). Cross linking was achieved by adding 1 ml 1% formaldehyde in PBS, dicing the tissue with scissors, and incubating at room temperature for 20 min. Samples were spun 5 min at 800g at 4°C and the supernatant discarded. Pellets were washed twice with 1ml 0.125M glycine in PBS, spun 5 min at 800g, and the supernatant discarded. Next, tissue was homogenized in 1ml hypotonic buffer (10mM KCl, 10mM Tris pH 8, 1.5mM MgCl2
) using a Dounce homogenizer with an A pestle. Samples were then incubated on ice for 15 min allowing the cells to swell, followed by the addition of 100μL of 10% NP-40 and spinning of samples 1min at 16,000g. The supernatant was discarded and 360μL of RIPA lysis buffer (50mM Tris, 150mM NaCl, 1% NP-40, 0.1% SDS, 5mM EDTA, 1mM EGTA) was added to the remaining nuclear pellet. Samples were then sonicated for 10 min on a high setting (30sec on/30sec off) using a Bioruptor sonicator (Diagenode, Sparta, NJ, USA). Samples were then spun 5 min at 16,000g and the top 350μL of the supernatant was added to 100μL of 50% Protein G Agarose bead slurry blocked with salmon sperm DNA (Upstate, Billerica, MA, USA) and 1mL of RIPA buffer. Samples were incubated on a rotator at 4°C for 1 hour, spun at 3,300g for 1min and transferred to new tubes as ChIP input samples. From ChIP input tubes 425μL samples were aliquoted to new tubes for incubation with 2.5μg of NRSF antibodies (H290, Santa Cruz) or IgG. Samples were incubated on a rotator at 4°C overnight, then spun at 16,000g for 10 min and the top 360μL of the supernatant was transferred to a tube containing 30μL of 50% Protein G Agarose beads. Samples were incubated on the rotator at 4°C for 1 hour and then spun at 3,300g for 1 minute. Supernatant was discarded and the beads were washed 5 times by adding 1mL of RIPA buffer, incubating on the rotator at 4°C for 5 min, spinning at 3,300g, and aspirating the supernatant. To 20μL aliquots of the original ChIP input samples and to each pellet, 65μL of 20% Chelex (Biorad, Hercules, CA, USA) in water was added. The tubes were briefly mixed with a vortex mixer and incubated in boiling water for 10 min. The samples were allowed to cool and spun at 16,000g for 2 min. The top 35μL of supernatant containing immunoprecipitated or input DNA was transferred to new tubes for analysis.
Polymerase Chain Reaction (PCR) on recovered DNA was performed using primers directed at the Crh gene (Forward –AGT TTG GGG AAG AC T TAG GAA GAG, Reverse – CTA TCC GAC AGA CAC AGA CAA GAC) or the Beta Actin gene (Forward – GAC TAC CTC ATG AAG ATC CTG ACC, Reverse – GAG ACT ACA ACT TAC CCA GGA AGG). Gotaq green (Promega, Madison, WI, USA) polymerase mix was used with 10μM of each primer and 1-4μL of DNA template per 20μL reaction. ChIP input DNA samples were used to adjust the amount of DNA used in each PCR to equivalent levels. After 30 cycles of PCR, products were run through gel electrophoresis along with samples of a quantitative DNA ladder (Bioline Hyperladder I). Ethidium bromide and UV light was used to visualize the DNA bands and images were captured. Image analysis was performed on blinded samples using ImageJ (NIH) software to produce a standard curve from the quantitative DNA ladder. Band intensities for the PCR products were quantified using the standard curve.
Statistical considerations and analyses
All analyses were conducted without the knowledge of treatment. The significance of differences between groups was set at 0.05. Differences in the maternal behavior of control and experimental dams were analyzed using repeated measures analysis of variance (RMANOVA), in which treatment and time were included as fixed effects and dam as a repeated (subject) effect, using SAS v.9.2 (SAS Institute Inc., Cary, NC, USA. 2007). In order to correct for potential litter effect, the significance of differences among groups were examined using a mixed model analysis of variance (ANOVA) with treatment as a fixed effect, and the dam as a random effect, using SAS